Chronic Constriction Injury of the Distal Infraorbital Nerve (DIoN-CCI) in Mice to Study Trigeminal Neuropathic Pain

Animals are treated and cared for according to the guidelines for pain research in conscious animals of the International Association for the Study of PAIN and in line with the Flemish and European regulations for animal research and the ARRIVE guidelines. The protocol is approved by the institutional Ethical Committee.

1. Animals

1. Use male and female C57BL/6J mice (Janvier, 10 weeks old at arrival).
2. House male and female mice separately in standard solid-bottom mice cages in a colony room with a humidity of 40%-60% and a room temperature (RT) of 21 ± 1 °C.
3. Provide water and food ad libitum.
4. Keep mice under a normal 12:12 h dark/light cycle (lights on at 08:00).

2. Surgery

1. Per mouse, prepare one piece of chromic gut ligature (6-0) approximately 6 cm long and place it in sterile saline to avoid drying and becoming stiff and brittle.
2. Anesthetize the mouse with ketamine/xylazine (75/15 mg/kg, intraperitoneal, injection volume 10 mL/kg). Check the depth of anesthesia by pinching the skin between the toes. Ensure that the mouse does not flex its leg. If necessary, wait until the animal is fully anesthetized and/or administer additional ketamine/xylazine.
   NOTE: Supplementary analgesics should not be administered to avoid pre-emptive analgesic effects that may interfere with the development of trigeminal neuropathic pain.
3. Gently shave the buccal hair between the whisker pad and the eye to make an incision of approximately 4 mm just rostral to the infraorbital foramen. Take care not to damage the whiskers, as this may affect behavioral testing.
4. Fix the head of the mouse in a stereotaxic frame or otherwise fixate the head. Place the mouse on a heated pad or take care to maintain body temperature otherwise.
5. Apply ointment on both eyes to avoid drying. Scrub the shaved head area with alcohol, and then with betadine. Place a surgical drape exposing the shaved head area.
6. Use a microscope for steps 2.7 to 2.14.
7. Make a 4 mm skin incision perpendicular to the mid-line approximately halfway between the edge of the whisker pad and the eye, just rostral to the infraorbital foramen, and centered around the line between the center of the eye and the center of the whisker pad.
8. Expose the IoN by bluntly separating the superficial connective tissue. Take care to minimize musculature damage and avoid the motor nerve fibers. Ensure that the trunk of the IoN (1-1.5 mm in diameter) is accessible approximately 3 mm deep between where it exits the skull and where it branches out to the whisker pad (Figure 1).
9. Using a rotating motion, slip the head of a hooked ligation aid under the IoN, taking care not to damage the nerve.
10. Place the chromic gut ligature through the hole on the tip of the ligation aid and retract the ligation aid so that the ligature remains under the IoN and both ends of the ligature are more or less equidistant from the IoN.
11. Tie a "slip knot" from the two ends of the ligature and slide the knot against the IoN. Ensure that the slip knot allows a smooth action so that the degree of constriction can be accurately controlled. Slide the knot further and constrict the IoN. Reduce the diameter of the nerve by just a noticeable amount⁴. Place a normal knot on top of the slit knot to prevent it from slipping.
12. Cut the ends of the ligature leaving approximately 1.5 mm of free ends to prevent the knot from becoming undone.
13. Perform the sham surgery following steps 2.2-2.8.
14. Close the skin incision using synthetic absorbable sutures (6-0) and allow the animal to recover on a heated pad or under an infrared heating lamp.

3. Behavioral testing

1. Acclimate the mouse to the housing conditions for at least 8 days before pre-operative testing.
2. Before pre-operative testing, habituate the mouse to the test procedure at least once daily for 3 days.
3. Conduct testing in normal lighting conditions. If necessary, provide background noise to minimize disturbances from outside noises.
4. Observation of face grooming behavior
   1. Carry a single mouse from the housing to the test room in a covered plastic cage without any bedding material. Avoid external stimulation while transporting the animals.
   2. Place the mouse in a covered, transparent plastic cage without bedding (l x w x h: 12 cm x 12 cm x 17 cm) in front of a video camera. Place a mirror to view the animal's face when its back is toward the camera.
   3. Record the mouse's behavior for 10 min. During recording, ensure that the experimenter is not present in the room.
   4. After recording the next animal, clean the observation cage.
   5. Have an observer who is blind to the experimental conditions of the mouse analyze the recorded behavior.
   6. Note each face grooming episode while analyzing the 10 min recording. Face grooming is movement patterns in which the animal brings its forepaws in contact with facial areas.
   7. Make a distinction between isolated face grooming and face grooming behaviors during body grooming⁹. If a sequence is not preceded or followed by body grooming, the episode is labeled isolated face grooming. Body grooming is defined as patterns of movement that bring paws, tongue, or incisors in contact with a body area other than the face or the forepaws. If body grooming is present before or after a face grooming sequence, the episode is labeled as face grooming during body grooming.
   8. Determine the number of face grooming episodes by applying a 4 s cut-off criterion. A time period between grooming actions of less than 4 s is defined as a pause within a single episode. A time period greater than 4 s is defined as a full interruption of grooming actions between two episodes.
5. Mechanical stimulation testing
   1. Carry mice in groups of up to 6 animals from the housing to the test room in a covered cage with bedding. Again, take care to avoid external stimulation.
   2. Place the mice one at a time on a table.
      1. Place the tail of the mouse in a soft silicone clamp and attach the clamp magnetically to a metal plate on the table. The silicone material prevents the tail from slipping from the clamp while minimizing pressure on the tail.
      2. Place a three-walled plastic holder (65 mm x 25 mm x 23 mm) over the animal so that only the head of the mouse protrudes from the container. The size of the holder allows for head and forepaw movements but prevents the animal from turning around inside it. Finally, place a weight on top of the holder to keep the holder in place (Figure 2).
   3. Use a graded series of four von Frey hairs. The force required to bend the hairs is 0.02 g, 0.16 g, 0.4 g and 1.0 g.
   4. Habituate the mice to the restrainer and reaching movements for 10 min. Every 30 s, make a reaching movement per animal.
   5. When the animal is in a relaxed state, slowly apply the lightest von Frey hair within the IoN territory near the center of the vibrissae until the von Frey hair bends. Ensure that the stimulation takes no more than 1 s.
   6. Score the animal response to the stimulation to fit into one of the following response categories.
      1. Give a score of 0 when there is no response.
      2. Give a score of 1 for detection, i.e., the mouse turns its head toward the stimulating object and then explores the stimulus object.
      3. Give a score of 2 for withdrawal reaction, i.e., the mouse turns the head gently away or pulls it quickly backward when the stimulation is applied; sometimes, a single face wipe ipsilateral to the stimulated area occurs.
      4. Give a score of 3 for attacking, i.e., the mouse attacks the stimulus object, making biting and/or grabbing movements.
      5. Give a score of 4 for asymmetric face grooming, i.e., the mouse exhibits an uninterrupted series of at least three face-wash strokes directed toward the stimulated facial area.
   7. For each mouse, apply von Frey hairs in an ascending order of intensity and randomly stimulate the ipsilateral and contralateral sides. Apply each stimulus intensity one time on each side.
   8. Calculate the mean score from the responses to the four von Frey hairs within each animal. Calculate separate scores for the ipsilateral and contralateral sides.